Malaria is a mosquito-borne disease that causes over 2.7 million deaths per year according to estimates by the World Health Organization (WHO). Malaria is a potentially fatal blood disease caused by a parasite that is transmitted to human and animal hosts by the Anopheles mosquito. There are four human parasites including Plasmodium falciparum (P. falciparum), Plasmodium vivax (P. vivax), Plasmodium malariae (P. malariae) and Plasmodium ovate (P. ovale) of which P. falciparum is responsible for most of the mortality in humans. P. falciparum is dangerous not only because it digests the red blood cell's hemoglobin, but also because it changes the adhesive properties of the cell it inhabits, which causes the cell to stick to the walls of blood vessels. This becomes dangerous when the infected blood cells stick to the blood vessels, obstructing blood flow.
The life cycle of the malaria parasite in a human or animal begins when an infected mosquito injects malaria sporozoites into a new host during a blood meal. The sporozoites travel to the liver, where they invade hepatocytes (liver cells) and undergo a replication cycle which leads to the release of thousands of merozoites into the blood stream which happens approximately two weeks later. Released merozoites then invade red blood cells and undergo an intraerythrocytic cycle of development. During the first 48 hours after infecting a red blood cell, a parasite goes through several phases of development. The first phase is the ring stage, in which the parasite begins to metabolize hemoglobin. The next phase is the trophozoite stage, during which the parasite metabolizes most of the hemoglobin, gets larger, and prepares to produce more parasites. Finally, the parasite divides asexually to form a multinucleated schizont. At the end of the cycle, the red blood cell bursts open and the released merozoites invade new red blood cells (see MicroWorlds™ electronic science magazine; Lawrence Berkeley National Laboratory, University of California). As the parasite matures inside the red blood cell, it modifies the adhesive properties of the cell it inhabits which becomes especially dangerous as these infected blood cells stick to the capillaries in the brain, obstructing blood flow, a condition called cerebral malaria. In addition, continuous rupturing of infected and uninfected blood red cells, the latter as a result of an immune-mediated mechanism, coupled to a reduction in the production of new red blood cells from the bone marrow (dyserythropoiesis) inevitably leads to anaemia.
Drug resistant malaria has become one of the most important problems in malaria control. Clinical resistance in vivo has been reported to all antimalarial drugs except artemisinin and its derivatives. The WHO recommendations for the treatment of drug resistant infections include the use of artemisinins in combination with other classes of antimalarials. However, the high cost of these drugs limits their accessibility to poor countries. In some parts of the world, artemisinin drugs constitute the first line of treatment, and are used indiscriminately as monotherapy for self treatment of suspected uncomplicated malaria, which in turn increases the risk of developing drug resistance. The problem of drug resistance can be attributed primarily to increased selection pressures on P. falciparum in particular, due to indiscriminate and incomplete drug use for self treatment. Resistance to chloroquine in P. falciparum, first reported in Thailand in 1961 is now widespread in most malaria endemic countries. Resistance to the antifolates pyrimethamine and cycloguanil, arose soon after their deployment as antimalarials. The addition of sulfa compounds created drug combinations that in many cases proved effective against the resistant parasites, however, resistance arose due to these combinations as well. Several mechanisms can account for changes in drug susceptibility in the malaria parasites, for example, physiological adaptations due to non genetic changes, selection of previously existing drug resistant parasites from a mixed population under drug pressure, spontaneous mutation, mutation of extranuclear genes, or the existence of plasmid-like factors.
Selection of mutants by the drugs themselves appears to be an important mechanism. In an environment where sub-therapeutic levels of the antimalarial drugs are present, those parasites which have resistance through their natural variation or through mutations clearly have an important biological advantage. This means that even though the resistant strains were initially in the minority, the continued drug mediated elimination of intra-specific competition from the non-resistant strains has allowed the resistant strains to attain numerical superiority, in fact, resistance to conventional antimalarial drugs such as chloroquine and sulfadoxine-pyrimethamine (SP) is widespread. Multidrug resistant P. falciparum malaria is highly prevalent in Southeast Asia, South America and Africa. Africa, which is the continent with the highest burden of the disease is also affected with increased mortality as a result (Roll Back Malaria, Facts on ACTs, WHO, January 2006). The majority of studies indicate that drug pressure selection is to blame for the emergence of resistant malaria. In genetically determined resistance, gametocytes from resistant parasite populations will be transmitted, promoting the spread of drug-resistant strains. Plasmodium parasites have extremely complex genomes, and the ease with which they can switch between the microenvironments in different hosts, and the metabolic changes required illustrates the difficulty in studying the exact modes of action of the antimalarial drugs on parasite metabolism (The Biology of Malaria Parasites: Report of a WHO Scientific Group. WHO Technical Report Series, 1987). Examples of antimalarials that are associated with drug-resistance are the antibiotic doxycycline, certain antifolates such as proguanil, pyrimethamine and sulfonamides, quinolines such as chloroquine, mefloquine and quinine, and naphthoquinones such as atovaquone.
Certain compounds such as 1,2-dihydrotriazines, 2,4-diaminopyrimidines and 2,4-diaminoquinazolines have been extensively studied as inhibitors of dihydrofolate reductase (DHFR), a key enzyme for maintaining pools of reduced folates. In the malarial parasites, DHFR exists as a part of the bifunctional enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS), and acts by reducing dihydrofolate to tetrahydrofolate, which is subsequently converted to 5,10-methylenetetrahydrofolate. This cofactor is utilized by thymidylate synthase (TS) to produce deoxythymidylate, a component of DNA which is essential for DNA synthesis and cell growth. Inhibition of DHFR results in inhibition of DNA synthesis and in parasite death. These inhibitors, also known as antifolates, are therefore potentially useful drugs against infectious agents, provided that they can selectively inhibit DHFR of the target parasites without substantially affecting the cells of the host.
Inhibitors of DHFR, generally known as antifolates or antifols, which have been shown to be effective antimalarials, include cycloguanil (Cyc), a 1,2-dihydrotriazine and pyrimethamine (Pyr), a 2,4-diaminopyrimidine, and derivatives thereof, with different substituents on positions 1 and 2 of the dihydrotriazine, and on positions 5 and 6 of the diaminopyrimidine. Some compounds in these classes are also good inhibitors of bacterial DHFR, and have antibacterial activity. Although cycloguanil, pyrimethamine and other described 1,2-dihydrotriazine, 2,4-diaminopyrimidine and 2,4-diaminoquinazoline derivatives are effective against wild type malaria parasites, they are not effective in vivo after oral administration against antifolate-resistant parasites, which have been shown to bear mutations in the DHFR and dihydropteroate synthase (DHPS). The degree of resistance generally increases with the number of DHFR mutations, prompting the need for novel drugs which are effective both against non-resistant and resistant strains of malaria parasites. Since the human host also has DHFR, these drugs must have selectivity for the parasite DHFR over the corresponding host enzyme, or inhibit it to a much lesser degree, as otherwise they may have toxicity to the host. Other characteristics of the drugs must also not lead to host toxicity. It is also preferable that these drugs do not have significant antibacterial activity, since they will have to be administered frequently in malaria endemic areas, to treat malaria re-infections, with the added danger of developing resistant strains of bacteria co-existing during the anti-malarial treatment.
Specific 1,2-dihydrotriazine derivatives such as WR99210 and its prodrug PS-15 are known to be effective inhibitors for some drug-resistant strains of malaria in vitro, although they also show toxicity in animal models (see Knight et al., Ann. Trop. Med. Parasitol. (1982) 76:1-7) and their manufacturing involves the production of a highly toxic by-product, TCDD, which must be strictly controlled to a ppb level. Several compounds related to PS-15 have been synthesized and some have been under clinical trials (Jensen et al., J. Med. Chem. (2001) 44:3925-31; U.S. Pat. No. 5,322,858 (1994); and Shearer et al., J. Med. Chem. (2005) 48:2805-2813). It was speculated that WR99210 binds to the DHFR, but the actual details of binding were unknown until a crystal structure of the enzyme-inhibitor complex was reported revealing molecular interactions between WR99210 and Plasmodium falciparum DHFR (Yuvaniyama et al., Nat. Struct. Biol. (2003) 10:357-65). Despite their potent in vitro anti-P. falciparum activity against non-resistant and antifolate resistant strains, it is unclear whether such compounds will ever be developed as an effective treatment against malaria as a consequence of their poor oral bioavailability.
Because of the increasing mortality associated with malaria, a need exists for better and more effective methods of disease control. In particular, a need exists for more effective treatment and prophylaxis against drug-resistant malaria, i.e., in the form of effective inhibitors of multiple mutant dihydrofolate reductase of Plasmodium falciparum. The present invention meets this and other needs.